Erythropoietin (Epo) is a glycoprotein hormone that serves as a major influence on red blood cell development. Previous analysis of transgenic mice with the human erythropoietin receptor (hEpoR) promoter driving the expression of beta-galactosidase, revealed staining of tissues of non-hematopoietic origin. Isolation of RNA from tissues that expressed this promoter construct also revealed the presence of endogenous mouse EpoR transcripts in these tissues, including the brain and primitive muscle. To further investigate the potential role of EpoR in brain, two cell lines of neuronal origin (NT2 and SHY5Y) were cultured and analyzed. Northern blot experiments revealed the presence of two bands which were positive when probed with both the full length EpoR and a 40-mer specific for hEpoR. PCR analysis of these bands indicate both a transcript of the predicted size containing 8 exons and a longer transcript containing IVS2 (the second intron of EpoR). These results support previous findings that in the processing of the nascent mRNA transcript, IVS2 is the final intron to be excised. The possible biological significance of these results is being analyzed. Further analysis of mouse brain tissue through RT-PCR suggest a possible alternate transcript. Currently, 5' RACE and sequencing techniques are being used to characterize the upstream regions of these brain EpoR transcripts. The EpoR reporter gene expression in day 12 embryos also mimicked that previously observed with the muscle-specific transcription factor myf-5, the earliest of four basic-helix-loop-helix regulatory proteins required for commitment of embryonic cells to myogenic development. The 5' untranslated transcribed region (5'UTR) of the human EpoR gene contains three myf-5 binding motifs (the mouse EpoR has two) and E- boxes (CANNTG). We find that the EpoR promoter has high activity in erythroid OCIM1 cells but little activity in promyoblast C2C12 cells. Addition of the E-boxes in the 5~UTR region activated transcription 60-fold in the C2C12 cell line, but had no effect in OCIM1 cells, suggesting that these myf-5 motifs may account for the some of the embryonic EpoR transgene expression in non-hematopoietic tissue and may direct EpoR expression during muscle cell development. When C2C12 cells are deprived of serum-rich media, differentiation is induced. In contrast, in the presence of Epo, differentiation is blocked and proliferation continues further suggesting a possible role for EpoR in the regulation of proliferation and differentiation in non- hematopoietic cells.